Evaluation of three alum-precipitated Aeromonas hydrophila vaccines administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella: immunokinetics, immersion challenge and histopathology / Avaliação de três vacinas de Aeromonas hydrophila precipitadas com alúmen administradas a Labeo rohita, Cirrhinus mrigala e Ctenopharyngodon idella: imunocinética, desafio de imersão e histopatologia

Aeromonas hydrophila is a cause of infectious disease outbreaks in carp species cultured in South Asian countries including Pakistan. This bacterium has gained resistance to a wide range of antibiotics and robust preventive measures are necessary to control its spread. No prior use of fish vaccines has been reported in Pakistan. The present study aims to develop and evaluate inactivated vaccines against local strain of A. hydrophila in Pakistan with alum-precipitate as adjuvant. The immunogenic potential of vaccine was evaluated in two Indian major carps (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) and a Chinese carp (Grass carp: Ctenopharyngodon idella). Fish were vaccinated intraperitoneally followed by a challenge through immersion. Fish with an average age of 4-5 months were randomly distributed in three vaccinated groups with three vaccine concentrations of 108, 109 and 1010 colony forming unit (CFU)/ml and a control group. Fixed dose of 0.1ml was applied to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected on 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using compliment fixation test (CFT). Fish were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/ml through immersion. Significantly higher levels of antibody titers were observed from 28 DPV in all vaccinated groups as compared to those in the control group. In challenge experiment the average RPS (relative percent survivability) was 71% for groups vaccinated with 109 and 1010 CFU/ml and 86% for 108 CFU/ml. Vaccine with 108 CFU/ml induced highest immune response followed by 109 and 1010 CFU/ml. The immune response of L. rohita and C. idella was better than that of C. mrigala. In general, normal histopathology was [...].

Aeromonas hydrophila é uma causa de surtos de doenças infecciosas em espécies de carpas cultivadas em países do sul da Ásia, incluindo o Paquistão. Essa bactéria ganhou resistência a uma ampla gama de antibióticos, e medidas preventivas robustas são necessárias para controlar sua disseminação. Nenhum uso anterior de vacinas para peixes foi relatado no Paquistão. O presente estudo tem como objetivo desenvolver e avaliar vacinas inativadas contra cepa local de A. hydrophila no Paquistão com precipitado de alúmen como adjuvante. O potencial imunogênico da vacina foi avaliado em duas carpas principais indianas (Rohu: Labeo rohita, Mori: Cirrhinus mrigala) e uma carpa chinesa (Grass Carp: Ctenopharyngodon idella). Os peixes foram vacinados por via intraperitoneal, seguido de um desafio por imersão. Peixes com idade média de 4-5 meses foram distribuídos aleatoriamente em três grupos vacinados com três concentrações de vacina de 108, 109 e 1010 unidades formadoras de colônias (UFC) / ml e um grupo de controle. Foi aplicada dose fixa de 0,1ml em cada peixe no 1º dia e dose de reforço 15 dias pós-vacinação (DPV). Amostras de sangue foram coletadas em 14, 28, 35, 48 e 60 DPV para determinar os títulos de anticorpos no soro sanguíneo usando o teste de fixação de elogio (CFT). Os peixes foram desafiados a 60 DPV com infecciosa A. hydrophila com 108 CFU / ml por imersão. Níveis significativamente mais elevados de títulos de anticorpos foram observados em 28 DPV em todos os grupos vacinados, em comparação com aqueles no grupo de controle. Na experiência de desafio, o RPS médio (sobrevivência percentual relativa) foi de 71% para os grupos vacinados com 109 e 1010 CFU / ml e 86% para 108 CFU / ml. A vacina com 108 UFC / ml induziu a maior resposta imune seguida por 109 e 1010 UFC / ml. A resposta imune de L. rohita e C. idella foi melhor do que a de C. mrigala. Em geral, histopatologia normal foi observada em diferentes [...].

Aeromonas sobria regulates proinflammatory immune response in mouse macrophages via activating the MAPK, AKT, and NF-κB pathways.

Aeromonas sobria, a Gram-negative bacterium that can colonize both humans and animals, is found in a variety of environments, including water, seafood, meat, and vegetables (Cahill, 1990; Galindo et al., 2004; Song et al., 2019). Aeromonas spp. are conditionally pathogenic bacteria in aquaculture, which can rapidly proliferate, causing disease and even death in fish, especially when the environment is degraded (Neamat-Allah et al., 2020, 2021a, 2021b). In developing countries, Aeromonas spp. have been associated with a wide spectrum of infections in humans, including gastroenteritis, wound infections, septicemia, and lung infections (San Joaquin and Pickett, 1988; Wang et al., 2009; Su et al., 2013). Infections caused by Aeromonas spp. are usually more severe in immunocompromised individuals (Miyamoto et al., 2017). The presence of a plasmid encoding a ß|-lactamase in A. sobria that confers resistance to ß-lactam antibiotics poses a huge challenge to the treatment of diseases caused by this microorganism (Lim and Hong, 2020). Consequently, an in-depth understanding of the interaction between A. sobria and its hosts is urgently required to enable the development of effective strategies for the treatment of A. sobria infections.

Molecular characterization and functional analysis of IL-18 in snakehead (Channa argus) during Aeromonas schubertii and Nocardia seriolae infections.

As a proinflammatory cytokine of the interleukin-1 (IL-1) family, IL-18 plays important roles in host protection against bacterial, viral, and fungal infection. We cloned the open reading frame of snakehead (Channa argus) IL-18 (shIL-18) and found that it contained 609 base pairs and encoded 202 amino acid residues. The shIL-18 included a conserved IL-1-like family signature and two potential IL-1ß-converting enzyme cutting sites; one was conserved in all analyzed IL-18s, but the other was unique to shIL-18. Unlike other IL-18s, shIL-18 also contained a predicted signal peptide. In this study, shIL-18 was constitutively expressed in all tested tissues, and its expression was induced by Aeromonas schubertii and Nocardia seriolae in the head kidney and spleen in vivo and by lipoteichoic acid, lipopolysaccharides, and polyinosinic-polycytidylic acid in head kidney leukocytes in vitro. Moreover, recombinant shIL-18 upregulated the expression of interferon-γ, IL-1ß, and tumor necrosis factor-α1 and -α2 and promoted the proliferation of leukocytes. Taken together, these results showed that IL-18 played crucial roles in host defense against bacterial infection in fish, as it does in mammals.

Pathogenic characterization of Aeromonas salmonicida subsp. masoucida turbot isolate from China.

Aeromonas salmonicida is a gram-negative bacterium that is the causative agent of furunculosis. An A. salmonicida strain was isolated from diseased turbot (Scophthalmus maximus) with the sign of furunculosis from North China. Based on vapA gene, the strain was further classified as A. salmonicida subsp. masoucida RZ6S-1. Culturing RZ6S-1 strain at high temperature (28°C) obtained the virulence attenuated strain RZ6S. Genome sequence comparison between the two strains revealed the loss of the type IV secretion system (T4SS) and type III secretion system (T3SS) from the native plasmid pAsmB-1 and pAsmC-1 of wild-type strain RZ6S-1, respectively. Further study demonstrated that the wild-type strain RZ6S-1, but not its derivative mutant RZ6S, can stimulate apoptosis. Elevated protein level of cleaved caspase-3 was detected from epithelioma papulosum cyprinid (EPC) cells infected with wild-type strain RZ6S-1 as compared with that infected with RZ6S strain. Meanwhile, the invasion of the mutant strain RZ6S was about 17-fold higher than the wild-type strain RZ6S-1, suggesting that some protein(s) from A. salmonicida subsp. masoucida RZ6S-1 suppress its invasion. The RZ6S mutant strain was attenuated, since its LD50 is over 10,000 times higher compared to the wild-type strain as revealed in the turbot infection model.

Aeromonas dhakensis is not a rare cause of Aeromonas bacteremia in Hiroshima, Japan.

Aeromonas dhakensis, a newly recognized species, is often misidentified as A. hydrophila, A. veronii, or A. caviae by commercial phenotypic tests. Limited data about A. dhakensis are available in Japan. We retrospectively analyzed the patients with monomicrobial Aeromonas bacteremia at Hiroshima University Hospital from January 2011 to December 2017, and species re-identification was conducted using rpoD and gyrB gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Of the 19 strains from blood isolates, A. caviae (n = 9, 47.4%), A. dhakensis (n = 4, 21.1%), A. hydrophila (n = 3, 15.8%), and A. veronii (n = 3, 15.8%) were re-identified. A. dhakensis was phenotypically misidentified as A. hydrophila (n = 3, 75%) or A. sobria (n = 1, 25%). A. dhakensis was also misidentified as A. caviae (n = 2, 50%), A. hydrophila (n = 1, 25%), and A. jandaei (n = 1, 25%) in MALDI-TOF MS system. Malignancies (n = 12, 63.2%) and liver cirrhosis (n = 7, 36.8%) were common comorbidities. Biliary tract infection was the most frequent source of Aeromonas bacteremia (n = 11, 57.9%). The major source of A. dhakensis bacteremia was also biliary tract infection (n = 3, 75%), and the 14-day infection-related mortality of A. dhakensis was 25%. A. dhakensis isolates showed similar clinical characteristics, antimicrobial susceptibility, and mortality with those of other Aeromonas species isolates. This study demonstrated that A. dhakensis is not a rare cause of Aeromonas bacteremia, but is often misidentified as A. hydrophila in Hiroshima, Japan. Further studies should be conducted to identify the geographical distribution and clinical impact of A. dhakensis in Japan.

Elevada frequência de Aeromonas spp. e genes de virulência em cultivos de tilápia-do-nilo (Oreochromis niloticus) em tanques-rede, na região semiárida de Pernambuco, Brasil / High frequency of Aeromonas spp. and virulence genes in Nile Tilapia farms (Oreochromis niloticus) in semi-arid regions of Pernambuco, Brazil

Objetivou-se avaliar a ocorrência de Aeromonas spp. em peixes e amostras de água na região semiárida de Pernambuco e avaliar a frequência de aerolissina (aerA), enterotoxina citotóxica (act), enterotoxina citotônica (alt) e serina protease (ahp) nesses isolados. Foram analisados 70 peixes vivos e oito mortos com sinais clínicos de aeromoniose e 16 amostras de água. Aeromonas spp. foram identificadas por análises microbiológicas (provas bioquímicas) e molecular, usando-se primers específicos para a região 16S rRNA, e a distribuição dos quatro fatores de virulência (aerA, alt, act e ahp) foi investigada por ensaio de PCR. Cento e cinquenta e cinco (84,7%) isolados foram confirmados como Aeromonas spp. na análise molecular. Os genes de virulência mais frequentes foram act (53,55%) e aerA (51,61%). De acordo com o tipo de amostra, observou-se maior frequência do gene aerA (87,5% P=0,0474) em isolados de peixes mortos e a menor frequência do gene act (47,73% P=0,0002) em peixes vivos. Este estudo demonstrou a presença de aeromoniose no cultivo de tilápias em tanques-rede, nos municípios de Jatobá e Petrolândia, na região semiárida de Pernambuco. A detecção de aerA, act e alt pode ser utilizada na tipagem de virulência de Aeromonas spp.(AU)

The purpose of this study was to evaluate the occurrence of Aeromonas spp. from fishes and tilapia net-cage farm water in semi-arid regions of Pernambuco and to evaluate the frequency of the aerolysin (aerA), cytotoxic enterotoxin (act), cytotonic enterotoxin (alt) and serine protease (ahp) genes in Aeromonas isolates. 70 live and eight dead fish with aeromoniosis clinical signs and 16 water samples were analyzed. Aeromonas spp. isolated were identified by microbiological (biochemical evidence) and molecular analysis using specific primers for 16SrRNA region, while the distribution of four virulence factors, including aerA, alt, act and ahp, was investigated by PCR assay. One hundred fifty-five (84.7%) isolates were confirmed as Aeromonas spp. by molecular analysis. The most frequent virulence genes in isolates were act (53.55%) and aerA (51,61%). According to the kind of sample, the higher frequency of aerA gene (87.5% P= 0.0474) was observed in isolates from dead fish and the lowest frequency of act gene (47.73% P= 0.0002) from live fish. This study found the presence of aeromoniosis on tilapia farming in net-cages on Jatobá and Petrolândia counties in the semiarid Pernambuco region. The detection of aerA, act and alt can be used for virulence typing of Aeromonas spp. isolates.(AU)

Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro.

Aeromonas sobria is a pathogen causing food-borne illness. In immunocompromised patients and the elderly, A. sobria can leave the intestinal tract, and this opportunistically leads to severe extraintestinal diseases including sepsis, peritonitis, and meningitis. To cause such extraintestinal diseases, A. sobria must pass through the intestinal epithelial barrier. The mechanism of such bacterial translocation has not been established. Herein we used intestinal (T84) cultured cells to investigate the effect of A. sobria serine protease (ASP) on junctional complexes that maintain the intercellular adhesion of the intestinal epithelium. When several A. sobria strains were inoculated into T84 monolayer grown on Transwell inserts, the strain with higher ASP production largely decreased the value of transepithelial electrical resistance exhibited by the T84 monolayer and markedly caused bacterial translocation from the apical surface into the basolateral side of T84 monolayer. Further experiments revealed that ASP acts on adherens junctions (AJs) and causes the destruction of both nectin-2 and afadin, which are protein components constituting AJs. Other studies have not revealed the bacterial pathogenic factors that cause the destruction of both nectin-2 and afadin, and our present results thus provide the first report that the bacterial extracellular protease ASP affects these molecules. We speculate that the destruction of nectin-2 and afadin by the action of ASP increases the ability of A. sobria to pass through intestinal epithelial tissue and contributes to the severity of pathological conditions.

Enterotype-based Analysis of Gut Microbiota along the Conventional Adenoma-Carcinoma Colorectal Cancer Pathway.

The dysbiosis of human gut microbiota is strongly associated with the development of colorectal cancer (CRC). The dysbiotic features of the transition from advanced polyp to early-stage CRC are largely unknown. We performed a 16S rRNA gene sequencing and enterotype-based gut microbiota analysis study. In addition to Bacteroides- and Prevotella-dominated enterotypes, we identified an Escherichia-dominated enterotype. We found that the dysbiotic features of CRC were dissimilar in overall samples and especially Escherichia-dominated enterotype. Besides a higher abundance of Fusobacterium, Enterococcus, and Aeromonas in all CRC faecal microbiota, we found that the most notable characteristic of CRC faecal microbiota was a decreased abundance of potential beneficial butyrate-producing bacteria. Notably, Oscillospira was depleted in the transition from advanced adenoma to stage 0 CRC, whereas Haemophilus was depleted in the transition from stage 0 to early-stage CRC. We further identified 7 different CAGs by analysing bacterial clusters. The abundance of microbiota in cluster 3 significantly increased in the CRC group, whereas that of cluster 5 decreased. The abundance of both cluster 5 and cluster 7 decreased in the Escherichia-dominated enterotype of the CRC group. We present the first enterotype-based faecal microbiota analysis. The gut microbiota of colorectal neoplasms can be influenced by its enterotype.

Genes de virulencia en Aeromonas spp. (Aeromonadales: Aeromonadaceae) aisladas de Oreochromis spp. (Perciformes: Cichlidae) para consumo humano en México / Virulence genes in Aeromonas spp. (Aeromonadales: Aeromonadaceae) isolated from Oreochromis spp. (Perciformes: Cichlidae) destined for human consumption in Mexico

Resumen Las especies del género Aeromonas se encuentran ampliamente distribuidas en ecosistemas acuáticos, son bacilos Gram negativas, oxidasa positivas y fermentadoras de glucosa que han sido consideradas patógenas emergentes en humanos. Por otra parte, Aeromonas pertenece a la microbiota normal de los peces, no obstante, estos microorganismos poseen una diversidad de factores de virulencia responsables de una variedad de infecciones en humanos, principalmente de tipo gastrointestinal. La presencia de Aeromonas en productos destinados a consumo de alta demanda comercial como la tilapia genera preocupación sanitaria por el potencial patogénico que posee esta bacteria. En este contexto, identificar genes de virulencia presentes en cepas de Aeromonas aisladas en Oreochromis spp. para consumo humano en Reynosa, Tamaulipas, México; es de importancia ante la escasez de estudios moleculares al respecto en la zona. En el presente estudio se analizó el potencial patogénico de 15 cepas de Aeromonas previamente identificadas molecularmente mediante PCR y secuenciación, procedentes de Oreochromis spp. Mediante PCR se analizaron seis genes de virulencia (alt, ast, aerA, hlyA, gcat y stx1) y las cepas utilizadas como control fueron: Aeromonas hydrophila subsp. hydrophila ATCC 7966, Aeromonas caviae 429865 INP, Escherichia coli O157:H7 y Escherichia coli K12. El 100 % (n = 15) de las cepas presentaron al menos un gen de virulencia, el gen aerA se detectó en 86.66 % de las cepas analizadas, mientras que los genes ast y stx1 no fueron identificados. Se encontró que las cepas de Aeromonas presentaban genes asociados en una misma cepa: aerA/gcat, alt/aerA, alt/ aerA/gcat/hlyA y alt/aerA/gcat, de los cuales aerA/gcat se observó con mayor frecuencia y principalmente en A. veronii, mientras que, A. hydrophila presentó el mayor número de asociaciones de genes de virulencia. Estos hallazgos indican que las cepas de Aeromonas aisladas en Oreochromis spp. tienen el potencial de causar enfermedades en humanos. Por lo tanto, es necesario proporcionar información sobre esta bacteria emergente, para tratar y controlar eficazmente cualquier posible evento epidemiológico causado por la misma.(AU)

Abstract The genus Aeromonas are widely distributed in aquatic ecosystems are Gram-negative rods, oxidase-positive, and glucose-fermenting, considered emerging pathogens in humans. Aeromonas belongs to the fish microbiota, these microorganisms have a diversity of virulence factors responsible for a variety of infections in humans mainly gastrointestinal diseases. The presence of Aeromonas in products intended for consumption with high commercial demand such as tilapia generates sanitary concern due to the pathogenic potential of this bacteria. In this context, identification of virulence genes in strains of Aeromonas isolated in Oreochromis spp. intended for human consumption in Reynosa, Tamaulipas, Mexico is important due to the lack of molecular studies in this geographical area. In the present study the pathogenic potential of 15 strains of Aeromonas (A. veronii, A. hydrophila and A. schubertii) from Oreochromis spp. for human consumption were analyzed. Through PCR six virulence genes were analyzed (alt, ast, aerA, hlyA, gcat and stx1) and the strains used as control were: Aeromonas hydrophila subsp. hydrophila ATCC 7966, Aeromonas caviae 429865 INP, Escherichia coli O157: H7 and Escherichia coli K12. El 100 % (n = 15) of the strains harbored at least one virulence gene, aerA gene was detected in 86.66 % of the analyzed strains, while ast and stx1 genes were not identified. Moreover, Aeromonas strains had associated genes in the same strain: aerA / gcat, alt / aerA, alt / aerA / gcat / hlyA and alt / aerA / gcat, of which aerA / gcat were observed mostly in A. veronii, while A. hydrophila had the highest associations. These findings indicate that the strains of Aeromonas isolated in Oreochromis spp. have the potential to cause human diseases, and therefore, this species used as food, could be a vehicle for infections caused by Aeromonas. It also allows to provide information on this emerging microorganism to effectively treat and control any epidemiological event caused by Aeromonas spp. in the future.(AU)

Characterization, expression, and functional analysis of the hepcidin gene from Brachymystax lenok.

Hepcidin, a cysteine-rich antimicrobial peptide, is an important effector molecule in the innate immune system. Recently, Brachymystax lenok has become to be a valuable cold-water fish in China, particularly as the wild resources are rapidly declining. In this study, the hepcidin gene of Brachymystax lenok (Blhepc) has been cloned. The 870-bp mRNA contains a coding sequence (CDS) of 267 bp that encodes 88 amino acid residues. Amino acid sequence identities of Blhepc with hepcidin in Oncorhynchus mykiss, Salmo salar, and Hucho taimen were found to be 93.18%, 89.77% and 93.18%, respectively. Phylogenetic analysis indicated that Blhepc was clustered in the family Salmonidae. The putative signal peptide and the mature peptide contained 24 and 25 amino acid residues, respectively. The RXXR motif for recruitment of propeptide convertase was identified upstream of the mature peptide of Blhepc by sequence analysis. The N-terminal amino acid residues of the mature Blhepc peptide were Q-SH-L, a structure involved in regulating iron metabolism. Eight conserved cysteine residues in the mature peptide were held together by four disulfide bonds. Expression profiling of Blhepc indicated its highest level in the liver; its expression was stronger in males than in similar-aged females. Moreover, its expression in the liver increased significantly with age. Expression of Blhepc in six immune tissues showed increase in various degrees when challenged with Aeromonas salmonicida and Aeromonas hydrophila. A synthetic Blhepc mature peptide was validated to have significant antimicrobial activity against gram-negative and gram-positive bacteria and fungi in vitro. These results show that Blhepc may be an important component in the innate immunity of Brachymystax lenok, which could provide antimicrobial activities against invading pathogens.

Protective potency of clove oil and its transcriptional down-regulation of Aeromonas sobria virulence genes in African catfish (Clarias gariepinus L.).

Disease episodes of fish caused by Aeromonas species are moved to the top list of limiting problems worldwide. The present study was planned to verify the in vitro antibacterial activities as well as the in vivo potential values of clove oil and ciprofloxacin against Aeromonas sobria in African catfish (Clarias gariepinus). The in vitro phenotypic virulence activities and the successful amplification of aerolysin and hemolysin genes in the precisely identified A. sobria strain were predictive for its virulence. In the in vivo assay, virulence of A. sobria strain was fully demonstrated based on constituent mRNA expression profile of tested virulence genes and typical septicemia associated with high mortalities of infected fish. Apparent lower mortality rates were correlated well with both decrescent bacterial burden and significant down-regulated transcripts of representative genes in the treated groups with clove oil, followed by ciprofloxacin as a prophylactic use for 15 days (P < 0.0001); however, the essential oil apart from ciprofloxacin significantly enhanced different hematological parameters (P < 0.05). In addition, administration of antibiotic may be considered as a pronounced stress factor in the fish even when it used in the prophylactic dose. In conclusion, medicinal plants-derived essential oils provide a virtually safer alternative to chemotherapeutics on fish, consumers and ecosystems.

Recuperación y diferenciación de aeromonas con CromoCen AE y CromoCen AGN / Recovery and differentiation of Aeromonas with CromoCen AE and CromoCen AGN

INTRODUCCIÓN: en la actualidad las especies del género Aeromonas han emergido como un problema de salud pública, son ellas los agentes etiológicos de las enfermedades diarreicas con el aumento de la atención médica por años. Los procedimientos convencionales para su diagnóstico son muy engorrosos, laboriosos y duraderos. Una nueva metodología que emplea medios de cultivo cromogénicos ha permitido la simplificación y aceleración de su diagnóstico, que ofrece resultados altamente específicos. OBJETIVO: estudiar el efecto de la combinación de diferentes agentes selectivos de los microorganismos grampositivos sobre el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. MÉTODOS: se estudió el efecto inhibidor de la combinación de agentes selectivos (desoxicolato de sodio (0,05-0,2 g·L-1), sales biliares (0,65 g·L-1), verde brillante (0,025-0,03 g·L-1), cristal violeta (0,001-0,01 g·L-1) y sulfito de sodio (0,8 g·L-1) sobre los microorganismos grampositivos, así como la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas. Como base se utilizó la formulación de CromoCen AGN, sin el desoxicolato de sodio. RESULTADOS: los valores de las productividades de los medios CromoCen AE y CromoCen AGN a partir del inóculo 1,5 × 102 UFC·mL-1 resultaron, para: A. hydrophila 116,8 % y 23,9 %, A. caviae 100,8 % y 3,95 %, A. bestiarium 93,6 % y 28,8 %, A. culicicola 85,1 % y 66,12 %, A. veronii 116,7 % y 59,2 %, A. popoffi 86,56 % y 13,2 %, A. trota 94,8 % y 11,25 % y para A. eucrinophila 103,9 % y 2,80 %. La nueva composición cromogénica logró la diferenciación de los microorganismos por sus características culturales: color, forma, superficie, bordes en las colonias y proteólisis del medio circundante. CONCLUSIONES: la combinación de diferentes agentes selectivos para la inhibición de los microorganismos grampositivos coadyuvo el aumento de la capacidad de recuperación, cuantificación y diferenciación de las especies de Aeromonas.

INTRODUCTION: Species of the genus Aeromonas are a current public health problem, for they are the etiological agents responsible for the growing incidence of diarrheal diseases requiring medical care. Conventional procedures for their diagnosis are very complicated, laborious and time-consuming. A new methodology based on the use of chromogenic culture media allows diagnostic simplification and acceleration, yielding highly specific results. OBJECTIVE: Study the effect of combining several selective agents for gram-positive microorganisms upon an increased capacity for recovery, quantification and differentiation of Aeromonas species. METHODS: Assessment was conducted of the inhibiting effect of combined selective agents (sodium deoxycholate (0.05-0.2 g·L-1), bile salts (0.65 g·L-1), brilliant green (0.025-0.03 g·L-1), crystal violet (0.001-0.01 g·L-1) and sodium sulfite (0.8 g·L-1)) on gram-positive microorganisms, as well as their capacity for recovery, quantification and differentiation of Aeromonas species. The base used was the CromoGen AGN formulation without sodium deoxycholate. RESULTS: Productivity values for the media CromoCen AE and CromoCen AGN based on inoculation of 1.5 × 102 CFU·mL-1 were 116.8 % and 23.9 % for A. hydrophila, 100.8 % and 3.95 % for A. caviae, 93.6 % and 28.8 % for A. bestiarium, 85.1 % and 66.12 % for A. culicicola, 116.7 % and 59.2 % for A. veronii, 86.56 % and 13.2 % for A. popoffi, 94.8 % and 11.25 % for A. trota, and 103.9 % and 2.8 0% for A. eucrinophila. The new chromogenic composition enabled differentiation of microorganisms based on their cultural characteristics: color, shape, surface, colony borders and environmental proteolysis. CONCLUSIONS: Combination of various selective agents for the inhibition of grampositive microorganisms led to an increased capacity for recovery, quantification and differentiation of Aeromonas species.

Ocorrência de Aeromonas spp. em alimentos de origem animal e sua importância em saúde pública / Occurence of Aeromonas spp. in foods of animal origin and its importance in public health

Aeromonas spp. são bactérias Gram negativas, oportunistas, de natureza ubíqua, isoladas principalmente de amostras de água. Até o presente momento foram reconhecidas 31 espécies, sendo as de maior importância médica Aeromonas hydrophila, Aeromonas caviae e Aeromonas veronii. A patogenicidade do gênero é considerada multifatorial, sendo este produtor de diversos tipos de toxinas e com envolvimento de outros fatores capazes de facilitar a penetração e o estabelecimento do agente no hospedeiro, causando doença. O objetivo desta revisão é elucidar o papel dos alimentos de origem animal como fontes de contaminação de bactérias do gênero Aeromonas para o ser humano. Isolamentos de aeromonas de diversos produtos de origem animal têm sido relatados, como carne, leite e seus derivados, além de frutos do mar, e em ambientes de processamento, como abatedouros, frigorífcos e laticínios. Tem-se buscado determinar fontes de contaminação dos alimentos, e a água foi definida como o principal disseminador. Aeromonas já foi definida como sendo a causadora de diversas enfermidades, desde afecções gastrointestinais até casos de meningite e morte. Considerando os alimentos de origem animal como importantes veículos de transmissão para o ser humano e o reconhecimento da água como fonte de disseminação do agente, torna-se imprescindível o tratamento adequado da água utilizada nos estabelecimentos processadores de alimentos para a segurança alimentar.(AU)

Aeromonas spp. are opportunistic, ubiquitous Gram negative bacteria, mostly isolated from water samples. Until the present time, 31 species have been recognized, and the most medically important are Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii. The pathogenicity of the genus is considered to be multifactorial, and it can produce several types of toxins, with the involvement of other factors that facilitate the penetration and the establishment of the agent in the host, thus causing the disease. Te objective of this review is to elucidate the role of foods of animal origin as sources of contamination of aeromonas to humans. Aeromonas have been reported in various animal products such as meat, milk, dairy products, and seafood, and also in processing environments such as slaughterhouses, meat and dairy plants. There has been the attempt to determine sources of food contamination, being the water defined as the main disseminator. Aeromonas has been defined as the cause of many diseases, from gastrointestinal affections to cases of meningitis and even death. Considering that animal foods are an important source of contamination for humans and because of the recognition of water as a source of dissemination, it is essential for food security to provide the proper treatment of water used in food processing establishments.(AU)

Molecular cloning and expression analysis of two ß-defensin genes in the blunt snout bream (Megalobrama amblycephala).

ß-Defensins are a group of cysteine-rich, cationic antimicrobial peptides that play important roles in innate immune system against pathogenic microbes invading. In this study, the part-length cDNA sequences of two ß-defensin genes (maΒD-1, maΒD-2) in blunt snout bream (Megalobrama amblycephala) were identified. Homology analysis showed that the cDNA sequences of maΒD-1 and maΒD-2 had high similarities to those in common carp and zebrafish. Real-time quantitative PCR results exhibited that expression level of maΒD-1 in juvenile tissues was the highest in skin, followed by blood and liver, whereas maΒD-2 was lowly expressed in liver, kidney, brain and foregut. In the early development period, fertilized eggs to 31-day post-hatching (dph) larvae, the expression levels of maΒD-1 were higher at the stage from heart beat stage to 3 dph with the highest value at 1 dph, whereas maΒD-2 was expressed higher at fertilized eggs and late cleavage stages. Following bacterial stimulation in vivo by Aeromonas sobria, maΒD-2 expressions were significantly up-regulated in liver, skin, gill, and foregut of juveniles, and maΒD-1 expressions were significantly up-regulated in liver and skin. The results suggest that maΒD-1 and maΒD-2 may play important roles in protecting blunt snout bream embryos, fry and juveniles from pathogenic microbe invading.

Flap infection associated with medicinal leeches in reconstructive surgery: two new drug-resistant organisms.

The use of medicinal leeches in reconstructive surgery has proven value for the salvage of flaps with venous congestion but is associated with a risk of leech-acquired infection. The most common leech-associated organism is Aeromonas hydrophila, which antibiotic prophylaxis is typically directed against. The authors describe two new multidrug-resistant organisms acquired from medicinal leech therapy that resulted in flap infection. The evaluation of suspected leech-borne infection and management protocol for this leech-acquired resistant multi-organism infection is presented.

Molecular characterization of Aeromonas spp. and Vibrio cholerae O1 isolated during a diarrhea outbreak / Caracterização molecular de Aeromonas spp. e Vibrio cholerae O1 isolados durante um surto de diarréia

This work aimed to assess pathogenic potential and clonal relatedness of Aeromonas sp. and Vibrio cholerae isolates recovered during a diarrhea outbreak in Brazil. Clinical and environmental isolates were investigated for the presence of known pathogenic genes and clonal relatedness was assessed by intergenic spacer region (ISR) 16S-23S amplification. Four Aeromonas genes (lip, exu, gcat, flaA/B) were found at high overall frequency in both clinical and environmental isolates although the lip gene was specifically absent from selected species. A fifth gene, aerA, was rarely found in A. caviae, the most abundant species. The ISR profile revealed high heterogeneity among the Aeromonas isolates and no correlation with species identification. In contrast, in all the V. cholerae isolates the four genes investigated (ctxA, tcpA, zot and ace) were amplified and revealed homogeneous ISR and RAPD profiles. Although Aeromonas isolates were the major enteric pathogen recovered, their ISR profiles are not compatible with a unique cause for the diarrhea events, while the clonal relationship clearly implicates V. cholerae in those cases from which it was isolated. These results reinforce the need for a better definition of the role of aeromonads in diarrhea and whether they benefit from co-infection with V. cholerae.

O objetivo deste trabalho foi estabelecer o potencial patogênico e a relação clonal de isolados de Aeromonas sp. e Vibrio cholerae obtidos durante um surto de diarréia. Isolados clínicos e ambientais foram investigados quanto à presença de genes de virulência e sua relação clonal foi obtida através de amplificação da Região Espaçadora Intergênica (REI) 16S-23S. Quatro genes de Aeromonas (lip, exu, gcat, flaA/B) foram encontrados em alta frequência embora o gene lip tenha se mostrado ausente em algumas espécies. Um quinto gene, aerA, foi raramente encontrado em A. caviae, a espécie mais abundante. O perfil da REI revelou alta heterogeneidade entre os isolados de Aeromonas e nenhuma correlação com espécie. Em contraste, todas as amostras de V. cholerae amplificaram os genes investigados (ctxA, tcpA, zot e ace) e revelaram perfil clonal através de REI e RAPD. Embora Aeromonas tenha sido o principal patógeno isolado, o perfil da REI não é compatível como única causa para os eventos de diarréia, enquanto a relação clonal de V. cholerae aponta esse microrganismo como o provável agente do surto. Estes resultados reforçam a necessidade de definir melhor o papel de Aeromonas em diarréias e de que forma essas bactérias se beneficiam quando em co-infecção com V. cholerae.

Characterization of pathogenic Aeromonas veronii bv. veronii associated with ulcerative syndrome from Chinese longsnout catfish (Leiocassis longirostris Günther)

273 bacterial strains were isolated from 20 Chinese longsnout catfish samples. The biochemical characteristics of all strains conformed to the species description of Aeromonas veronii bv. veronii on the basis of Vitek GNI+ card. Furthermore, 16S rDNA, gyrB and rpoD sequences of the representative strain PY50 were sequenced and showed high similarity with A. veronii bv. veronii in Genbank. Antibiotic-resistance of the representative strain PY50 was assessed by the Kirby-Bauer disk diffusion method, and the results showed it was susceptible and moderately susceptible to 13 and 4 of the 21 antimicrobial agents tested. Extracellular products of strain PY50 contained gelatinase, lecithinase, elastase, most of lipase and lipopolysaccharide. Virulence of strain PY50 and extracellular products to Chinese longsnout catfish were also tested, and LD50 were about 3.47~10(4) CFU per fish and 11.22 ƒÊg per fish in intraperitoneal injection respectively. This is the first report that A. veronii bv. veronii was the pathogenic agent of ulcerative syndrome in Chinese longsnout catfish.

Aeromonas spp.-mediated cell-contact cytotoxicity is associated with the presence of type III secretion system.

In the study we examined the production of cytotonic and cytotoxic toxins and the presence of a type III secretion system (TTSS) in 64 Aeromonas spp. strains isolated from fecal specimens of patients with gastroenteritis. We observed that contact of the bacteria with host epithelial cells is a prerequisite for their cytotoxicity at 3 h incubation. Cell-contact cytotoxic activity of the strains was strongly associated with the presence of the TTSS. Culture supernatants of the strains induced low cytotoxicity effects at the same time of incubation. Cell-free supernatants of 61 (95%) isolates expressed cytotoxic activity which caused the destruction of HEp-2 cells at 24 h. Moreover, 44% strains were cytotonic towards CHO cells and 46% of strains invaded epithelial cells.

Clinical characteristics of seven patients with Aeromonas septicemia in a Japanese hospital.

The genus Aeromonas comprises flagellated gram-negative rods widely distributed in freshwater, estuarine and marine environments. Aeromonas species may cause a variety of illnesses in humans, such as enterocolitis and septicemia, especially in warmer tropical or subtropical environments. To recognize the characteristics of Aeromonas septicemia in Japan, we reviewed laboratory data and medical records in our hospital. During 11 years (from 2000 to 2010), Aeromonas septicemia was observed in seven patients involving six female subjects. Six patients were observed in summer or fall. The incidence of Aeromonas septicemia was about 0.07 per 1000 admissions, and two out of the seven patients died. All patients had underlying diseases such as malignancy (six patients) and choledocholithiasis (one patient). Two patients developed septicemia within two days after ingesting raw seafood. Five patients developed Aeromonas septicemia > 48 h after admission. Fever was present in all patients, and four out of the seven patients developed septic shock. All patients developed monomicrobial septicemia. A. hydrophila was isolated from five patients, and A. caviae and A. veronii biovar sobria were isolated from one patient each. Most antimicrobial agents had high activity against the isolated strains. However, a carbapenem-resistant strain appeared in one patient during treatment and led to death. Aeromonas septicemia is uncommon in temperate areas but can occur particularly in warm seasons. Immunocompromised conditions and recent ingestion of raw fish or shellfish are important characteristics of developing Aeromonas septicemia.